A variety of molecular biology techniques can be used to detect RNAs. Hybridization of nucleic acid probes to complementary RNA after electrophoretic separation is used extensively in the analysis of gene structure, expression and diagnostic tests (Fleming, Journal of Pathology, 166(2), 95–6, 1992; Singer et al, Blood, 74(6), 2295–301, 1989). These techniques require technically demanding generation of the antisense RNA probe, and denaturing gels that may contain harmful chemicals such as formaldehyde. Additionally these methods do not allow the precise cellular localization of the RNA target. In situ hybridization (ISH) permits the localization of specific nucleic acid sequences at the cellular level and detection of gene expression frequencies. However, the method though highly specific may often be overshadowed by a relatively low detection sensitivity (Chen et al., Transplantation, 55(1), 146–53, 1993; Martinez et al., J. Hist. & Cytochem., 43(8), 739–47, 1995).
Amplification of the target mRNAs by PCR is used to improve sensitivity enabling detection of low abundancy mRNAs in a given cell (Chen et al., Transplantation, 55(1), 146–53, 1993; Tecott et al, Science, 240(4859), 1661–4 1988; Nuovo et al., A. J. Path., 139(6), 1239–44, 1991; Bagasra et al., New Engl. J. Med., 326(21), 1385–91, 1992; Patterson et al., Science, 260(5110), 976–9, 1993; Staecker et al., Biotechniques, 16(1), 1994; Chiu. et al., Journal of Histochemistry & Cytochemistry, 40(3), 333–41, 1992; Heniford, et al., Nucleic Acids Research, 21(14), 3159–66, 1993; Nuovo et al., Genome Research, 2(2), 117–23, 1992; Peters et al., American Journal of Pathology, 150(2), 469–76, 1997; Tolker-Nielsen et al., Molecular Microbiology, 27(6), 1099–105; 1998; Lee et al., Laboratory Investigation, 78(4), 453–9, 1998). Various detection methods have been used following target mRNA amplification in tissue sections or cell suspensions (Chen et al., Transplantation, 55(1), 146–53, 1993; Nuovo et al., A. J. Path., 139(6), 1239–44, 1991; Staecker et al., Biotechniques, 16(1), 1994; Peters et al., American Journal of Pathology, 150(2), 469–76, 1997; Tolker-Nielsen et al., Molecular Microbiology, 27(6), 1099–105; 1998; Lee et al., Laboratory Investigation, 78(4), 453–9, 1998; Patel, et al., American Journal of Pathology, 144(1), 7–14; 1994; Haase, et al., Proc. Nat'l Acad. Sci. USA, 87(13), 4971–5, 1990; Negro et al., Proc. Nat'l Acad. Sci. USA, 89(6), 2247–51, 1992).
RT-PCR followed by a separate ISH step using either a radiolabeled or non-isotopically labeled complementary probe for detection and localization of the target mRNAs is quite cumbersome (Mee, et al., J. Path., 182(1), 22–8, 1997; Ertsey and Scavo, Biotechniques, 24(1), 92, 94, 96, 98–100, 1998).
Alternatively, direct incorporation of a labeled deoxynucleotide in the amplification step can be used (Chen et al., Transplantation, 55(1), 146–53, 1993; Martinez et al., J. Hist. & Cytochem., 43(8), 739–47, 1995; Heniford, et al., Nucleic Acids Research, 21(14), 3159–66, 1993; Patel, et al., American Journal of Pathology, 144(1), 7–14; 1994; Boshoff et al., Nature Medicine, 1(12) 1274–8, 1995). However, direct incorporation of the labeled dNTP results in a high background from nonspecific amplification following mis-printing (extension of primers annealed to non-target sequences) (O'Leary et al., Journal of Pathology, 178(1), 11–20, 1996; Long et al., Histochemistry, 99(2), 151–62, 1993). Hot-start PCR (Nuovo, PCR in situ Hybridization: Protocols and Applications. Lippincott-Raven, Philadelphia, pp. 54–99, 1996) and the use of labeled primers in PCR (Embleton et al., Nucleic Acids Research, 20(15), 3831–7, 1992) instead of direct incorporation of labeled deoxynucleotide helps in eliminating background due to mis-printing or due to primer-independent events. The use of labeled primers across the intron-exon junctions may also circumvent the problems of non-specific amplification from genomic DNA (Lee et al., Laboratory Investigation, 78(4), 453–9, 1998; Kuo et al., Biochem. & Mol. Biol. Intern'l, 40(1), 119–25, 1996). In spite of DNase I pretreatment of the samples (Martinez et al., J. Hist. & Cytochem., 43(8), 739–47, 1995; Tolker-Nielsen et al., Molecular Microbiology, 27(6), 1099–105; 1998; Ertsey and Scavo, Biotechniques, 24(1), 92, 94, 96, 98–100, 1998; Nuovo, Meth. Mol. Biol., 33:223–41, 1994) and hot-start modification (Nuovo, PCR in situ Hybridization: Protocols and Applications. Lippincott-Raven, Philadelphia, pp. 54–99, 1996), non-specific amplification from undigested genomic DNA still remains a severe drawback in presently available methods (Komminoth et al., Path. Res. & Pract., 190(11), 1017–25, 1994), especially in cases where genomic sequence data is incomplete.